PROJECT SUMMARY Over 25 million Americans have asthma, which places an economic burden on the U.S. of $81.9 billion. Airway mucus overproduction is a hallmark of asthma, and the quantity and tenacity of mucus are increased during airway exacerbations. Mucolytics degrade mucus, but do not reduce its production. Although corticosteroids and other anti-inflammatory therapies indirectly decrease mucus production, and anti-cholinergics inhibit mucus release from airway epithelia, a significant number of asthmatic continue to suffer from asthma exacerbations. Clear unmet medical need exists for new therapeutics that directly target airway mucus overproduction. Airway inflammation and excess mucins activate inositol requiring enzyme 1 (IRE1), a transmembrane endoplasmic reticulum protein that contains cytoplasmic kinase and RNase domains. IRE1 exists in two isoforms, ? and ?. IRE1? is ubiquitously expressed, but IRE1? is only expressed in mucous cells of the respiratory and GI tracts. Key residues in the kinase ligand binding pocket differ for the two isozymes. Activation of IRE1? (but not IRE1?) kinase and RNase is required for airway mucin production. IRE1? (but not IRE1?) expression is up-regulated in asthmatic human bronchial epithelia (HBE), providing an amplifying mechanism for airway exacerbations due to mucus overproduction. Irex Pharma has developed novel IRE1?- specific assays to support a full synthetic medicinal chemistry campaign to identify potent, selective and efficacious IRE1? inhibitors to treat airway mucus overproduction. Our focused testing of IRE1 ligands revealed a distinct mechanism for blocking IRE1?-dependent mucin production. Most IRE1 kinase inhibitors tested activate the IRE1 RNase. We identified an IRE1? ligand that blocks both kinase and RNase activities and mucin production in HBE, providing proof-of-concept that a small molecule IRE1? ligand with this profile can block mucus overproduction in human airways. Aim 1 proposes to use structure-based design to guide the synthesis of novel analogs based on our current hits. Structure-activity relationships will be established using an iterative approach. Our goal is to identify nontoxic, patentable, nanomolar compound(s) using our team's extensive knowledge of kinase inhibitor design, computational chemistry, synthetic chemistry, and novel IRE1 assays. Aim 2 proposes to test our IRE1? inhibitors with favorable profiles for ability to reduce 1) interleukin-13 (IL-13)-stimulated MUC5AC production in HBE cultures and 2) house dust mite (HDM; an in vivo stimulus relevant to asthma)-induced airway mucin overproduction and hyper-responsiveness in mice. PK, early ADMET and kinase selectivity will be obtained for top compounds. Our goal is to identify a patentable IRE1? inhibitor able to reduce IL-13-increased MUC5AC in HBE and able to lower HDM-induced MUC5AC production and hyper-responsiveness in mouse airways with favorable potency, selectivity, toxicity and developability profiles. Completion of these studies will lay the groundwork for Phase II development of IRE1? antagonists as novel therapeutics for asthma.